Recently, a large number of measurements using intermolecular interactions such as immune responses are being carried out in clinical tests, etc. However, since conventional methods require complicated operations or labeling substances, several techniques are used that are capable of detecting the change in the binding amount of a test substance with high sensitivity without using such labeling substances. Examples of such a technique may include a surface plasmon resonance (SPR) measurement technique, a quartz crystal microbalance (QCM) measurement technique, and a measurement technique of using functional surfaces ranging from gold colloid particles to ultra-fine particles. The SPR measurement technique is a method of measuring changes in the refractive index near an organic functional film attached to the metal film of a chip by measuring a peak shift in the wavelength of reflected light, or changes in amounts of reflected light in a certain wavelength, so as to detect adsorption and desorption occurring near the surface. The OCM measurement technique is a technique of detecting adsorbed or desorbed mass at the ng level, using a change in frequency of a crystal due to adsorption or desorption of a substance on gold electrodes of a quartz crystal (device). In addition, the ultra-fine particle surface (nm level) of gold is functionalized, and physiologically active substances are immobilized thereon. Thus, a reaction to recognize specificity among physiologically active substances is carried out, thereby detecting a substance associated with a living organism from sedimentation of gold fine particles or sequences.
In all of the above-described techniques, the surface where a physiologically active substance is immobilized is important. Surface plasmon resonance (SPR), which is most commonly used in this technical field, will be described below as an example.
A commonly used measurement chip comprises a transparent substrate (e.g., glass), an evaporated metal film, and a thin film having thereon a functional group capable of immobilizing a physiologically active substance. The measurement chip immobilizes the physiologically active substance on the metal surface via the functional group. A specific binding reaction between the physiological active substance and a test substance is measured, so as to analyze an interaction between biomolecules.
As a thin film having a functional group capable of immobilizing a physiologically active substance, there has been reported a measurement chip where a physiologically active substance is immobilized by using a functional group binding to metal, a linker with a chain length of 10 or more atoms, and a compound having a functional group capable of binding to the physiologically active substance (Japanese Patent No. 2815120). Moreover, a measurement chip comprising a metal film and a plasma-polymerized film formed on the metal film has been reported (Japanese Patent Laid-Open No. 9-264843).
With regard to the aforementioned chip, the original activity of a physiologically active substance may be decreased by immobilization of the substance on a surface. Thus, only physiologically active substances that are strong against immobilization have been used as test materials. In addition, it has been pointed out that an excessive amount of carboxylic acid exists on the surface and that a physiologically active substance becomes inactivated due to the effects of such carboxylic acid. Under such circumstances, a surface capable of stably immobilizing all types of physiologically active substances has been desired for examining the relationship with enzyme activity and the like.
When a specific binding reaction between a physiologically active substance and a test substance is measured, the test substance is not necessarily comprised of a single component. There may also be a case where a test substance is required to be measured in a heterogeneous system such as a cell extract. In such a case, if contaminants such as various proteins or lipids are adsorbed on the detection surface nonspecifically, measurement/detection sensitivity is significantly reduced. The fact that nonspecific adsorption is highly likely to occur on the above detection surface has been problematic. In order to solve such problems, several methods have been studied. For example, a method of immobilizing a hydrophilic hydrogel on a metal surface via a linker, so as to repress physical adsorption, has been used (Japanese Patent No. 2815120, U.S. Pat. No. 5,436,161, and Japanese Patent Laid-Open No. 8-193948). However, nonspecific adsorption has not been sufficiently controlled by this method.
On the other hand, with regard to the aforementioned biosensor, in order to eliminate the influence of disturbance on measurement (e.g. temperature change, concentration change, or pressure change) so as to reduce baseline fluctuation, it is preferable that a measurement unit for measuring the specific binding reaction between a physiologically active substance and a test substance and a reference unit that does not carry out such a binding reaction exist on a single plane and be as adjacent as possible to each other. Thus, it becomes necessary that a reference unit coexist with a measurement unit on the surface of an SPR sensor of a thin polymer film.
U.S. Pat. No.6,444,254 describes a method for micro-stamping a biological ligand on the surface of a polymer, which comprises; forming on the polymer surface a first functional group by a method selected from the group consisting of hydrolysis, reduction, photoinduced graft polymerization, amination, polyethylene oxide surface cross polymerization, the chemical reaction of a terminal hydroxyl group, corona discharge, plasma etching, lasing, and ion beam treatment; allowing a stamp, on which a biological ligand having at least a second functional group is adsorbed, to come into contact with the surface of the first functional group, so as to form a covalent bond between the biological ligand and the first functional group on the polymer surface; and then separating the stamp from the polymer surface, so as to directly immobilize the biochemical ligand on the polymer surface via such a covalent bond. In the aforementioned method, a solid (PDMS) is allowed to come into contact with a polymer film for patterning. However, since a sensor used for SPR has a surface formed by attaching a thin polymer film on a thin metal film, it has little physical strength. Contact with a solid injures the surface of the sensor, and thus, the aforementioned method is not suitable for SPR.